Macrophages play an essential role in innate immunity. Optimal activation of macrophages occurs in two steps; a priming step provided for example by IFN-g; and subsequent stimulation by for example, bacteria or their products and modified matrix. Components such as hyaluronan (HA). Inflammatory responses are typically self- limiting but, the regulatory mechanisms involved in limiting of the response remain unclear. Although resolution of an inflammatory response may be partly due to elimination or dilution of the eliciting agent, evidence indicates that active regulatory mechanisms are also important. In addition to their pro- inflammatory functions, cytokines also can play a negative regulatory role in an inflammatory response. We established that pre-exposure to TNF markedly inhibited IFN-gamma priming of enhanced IL-12 production in HA or LPS stimulated murine macrophages. We propose to determine the cellular and molecular mechanisms that are involved in this novel cytokine regulatory pathway. Moreover, we will test the hypothesis that the anti- inflammatory functions of TNF observed in vivo involve its capacity to inhibit IFN-gamma priming of macrophage pro-inflammatory cytokine production. We will determine if TNF inhibits IFN-gamma priming for enhanced production of pro-inflammatory cytokines other than IL-12 including, IL-6, GM-CSF, IL-1 and IL-18 in vitro and in vivo. We will then determine if a) TNF interferes with IFN-induced signal transduction; or b) induces endogenous mediators known to downregulate production of IL-12 and other inflammatory cytokines, such as IL-1O, IL-4, TGFbeta, and PGE2 and that these factors mediate TNF inhibition of IFN-gamma primed cytokine production. We will determine if TNF inhibition of IFN-gamma priming occurs at the level of transCription or post-transcriptionally. The role of IL-12 p4O trans-acting factors (NF-kB, ets-2, IRF-1, cRel, CEBP and a novel 109 kDa protein) will be examined using electrophoretic mobility shift assays. The Cis-acting elements involved will be mapped by transient transfection of deletion mutants of a IL-12 p4O promoter-luciferase construct into macrophage cell lines. Finally, we will test the hypothesis that the anti-inflammatory functions of TNF observed in vivo involve its capacity to inhibit IFN-gamma priming of macrophage cytokine production. TNF-deficient mice treated with C. parvum exhibit a delayed but exaggerated inflammatory response in liver and spleen leading to death. We will compare levels of IL-12 and other cytokines in c. parvum and LPS challenged TNF-deficient and wild-type mice. The role of IL-12 or IL-1, IL-6, GM-CSF, IL-18 and IFN-gamma in the exaggerated granulomatous response to C. parvum will be examined by treating mice with neutralizing anti-cytokine antibodies. These studies will provide insight into the mechanisms responsible for regulating innate immune responses and will allow the rational design of new interventions for chronic inflammatory diseases.